Brassica plants resistant to disease

ABSTRACT

The present invention relates to novel  Brassica oleracea  plants resistant to  Albugo candida  and the seeds of said plants. The invention also relates to methods of making such plants and for producing seeds thereof. The invention further relates to molecular markers and use thereof in marker assisted breeding and for identifying  Albugo candida  resistance trait in  Brassica oleracea  plants.

This application is a 371 of International Application No.PCT/EP2010/063761 filed Sep. 19, 2010, which claims priority to EP09171021.0 filed Sep. 22, 2009, the contents of which are incorporatedherein by reference.

The present invention relates to novel plants resistant to Albugocandida and the seeds of said plants. The invention also relates tomethods of making such plants and for producing seeds thereof. Theinvention further relates to markers and use thereof in marker assistedbreeding and for identifying Albugo candida resistance trait.

Albugo candida (syn.: A. cruciferum also called white rust) or Albugocandida occurs in all parts of the world where cruciferous crops aregrown.

Albugo candida is a widespread disease that causes serious problems inmany Brassica growing areas.

White rust most commonly occurs on: field mustard (Brassica campestrisL.), leaf or Chinese mustard (B. juncea Zerj, & Coss.), black mustard(B. nigra (L.) Koch), broccoli (B. oleracea L. var. italica L.),cauliflower (B. oleracea L. var. botrytis L.), cabbage (B. oleracea L.var. capitata L.), Brussels sprouts (B. oleracea L. var. gemmifera DC),Chinese or celery cabbage (B. pekinensis (Lour.) Rupr.), rutabaga (B.campestris L. var. napoBrassica (L.) DC.), pak-choi (B. chinensis L.),turnip (B. rapa L.), radish (Raphanus sativus L.), and daikon (R.sativus L. var. longipinnatus Bailey).

Sporangia are produced in pustules and once liberated, are dispersed bywind, rain, or insects to neighbouring plants. The sporangia requiresome drying in order to germinate well. Each germinating sporangiumgives rise to five to seven zoospores. The preferred temperature forgermination ranges from 1 to 18° C., but is optimum between 10 and 14°C. Temperatures should be between 16 and 25° C., with the optimum at 20°C. for zoospores to produce germ tubes and penetrate plant tissue. Themoisture necessary for zoospore activity is ideal when in the form ofheavy dew or fog or during periods of extended rainfall and lowertemperatures. (Vanterpool, T. C. 1959. Oospore germination in Albugocandida. Can. J. Bot. 37:169-172).). Although A. candida showsspecialization to specific hosts, the disease seems to develop undersimilar conditions over a wide range of isolates and hosts (Gilijamse etal., 2004, Spencer, Philips and Jeger (eds.), Advances in Downy mildewResearch, Vol. 2, p. 107-118).

In contrast to other Brassica crops, oospores seem not to play asignificant role in the epidemiology of B. oleracea crops as no oosporeshave been reported yet. Asexual reproduction and survival of sporangiaon year-round production of B. oleracea crops does not necessitateoospores to be formed for survival during crop free periods. (Gilijamseet al., 2004, Spencer, Philips and Jeger (eds.), Advances in Downymildew Research, Vol. 2, p. 107-118).

Santos screened more than 30 Brassica oleracea accessions for resistanceto Albugo candida disease (Proc. Int. Symp. Brassica s. Ninth CruciferGenetics Workshop. Acta Hort. 407. ISHS 1996). His conclusions are thatthe majority of accessions showed susceptibility and among the testedaccessions, one or two wild species of Brassica oleracea var. costadacould consist in a source of resistance to explore and improve. In 2004Santos and Dias screened a core collection of 400 accessionsrepresenting the genetic and geographic diversity of B. oleracea. Againa great variability of reactions was found between and within accessionsof the core collection. Nine accessions presented 50-78% of resistantseedlings and could be considered as potential sources for breedingprograms for white rust resistance (Genetic Resources and Crop Evolution51: 713-722, 2004).

Pest management involves preventive and curative measures includingchemical treatment. Plowing or disking diseased plants and plant partsresults in rapid decomposition of infected tissues and helps tosignificantly reduce future white rust infection. Crop rotation withnoncruciferous host plants is also effective. Weed control and othersanitary methods are necessary too.

Resistance has been studied and successfully deployed with mustard andrutabaga, however, with cultivated Brassica oleracea species likebroccoli, white cabbage, Brussels sprouts and the like, such resistancehas not yet been identified.

The development of the acylalanine fungicide metalaxyl (Ridomil®)improved the ability to control while rust with fungicide application.Metalaxyl provides limited curative activity and some control ofsystemic infection.

Applications should be made to the soil and subsequently applied to thefoliage. Frequency of application would vary according to the length ofcrop and amount of rainfall experienced. In temperate environments asoil application and a minimum of 1-2 foliar applications during thecrop cycle is suggested. In the field, this fungus is difficult tocontrol because it causes infections that remain dormant in the fieldand develop into fruit decay during post-harvest storage. Crop losses ofup to 50% are not uncommon. Strategies for controlling Albugo candidaare limited by the emergence of strains that are resistant to one orseveral groups of fungicides. Most fungicides are protective in theiraction and will not suppress an established infection, which limitseffective control to pre-harvest applications of fungicide.

Therefore, good genetic resistance of the crop is important for itsprotection against the disease.

There was therefore a long felt and unmet need for convenient, efficientand economically sustainable strategies to protect Brassica species,especially cultivated Brassica oleracea species against Albugo candidainfestation. Therefore, there is an unfulfilled need for B. oleraceacultivated plants with an improved resistance to Albugo candida, whereinthe resistance is also easy to breed and transfer to commercialBrassica, particularly Brassica oleracea lines.

The present invention addresses this need by providing a Brassica plant,particularly a Brassica oleracea plant, and more particularly acultivated Brassica oleracea plant, which is resistant to Albugo candidaand thus protected from damage caused by this pathogen. The provision ofAlbugo candida resistant Brassica plants, especially Brassica oleraceaplants, is an environmentally friendly alternative for the use ofpesticides and may increase the efficiency of biological control optionsand contribute to successful integrated pest management programs.

The technical problem underlying the present invention is, therefore,the provision of an Albugo candida resistant cultivated Brassica plant,particularly a cultivated Brassica oleracea plant, which showsresistance to this pathogen.

The technical problem is solved by the provision of the embodimentscharacterized in the claims. Moreover, it was now found within the scopeof the present invention that the linkage between genes responsible forundesired, morphological changes at the plant and the gene responsiblefor the resistance to Albugo candida as present in the wild-type sourcematerial, could be broken and is, therefore, no longer present in thecultivated Brassica oleracea plant according to the invention.

Accordingly, the present invention addresses the problem ofunsatisfactory resistance to the disease Albugo candida in cultivatedBrassica oleracea. To achieve improved resistance to the disease incultivated Brassica oleracea, the present invention discloses thetransfer of a genetic determinant responsible for monogenicsemi-dominant-resistance to Albugo candida from a wild sourceundomesticated Brassica species, such as Kale cabbage for example todifferent cultivated B. oleracea species, such as white cabbage,broccoli, cauliflower and Brussels sprouts for example.

The resistance to Albugo candida can be transferred by means ofhybridization, followed by repeated backcrosses and disease tests in allbackcross generations. High level of resistance to Albugo candida isobtained in the resulting B. oleracea cultivated plants. The resistanceis stable, and can be transmitted to further generations and transferredto susceptible or less resistant B. oleracea cultivated plants.

Thus, the present invention discloses Brassica oleracea cultivatedplants resistant to Albugo candida, wherein the resistance to Albugocandida is monogenic and semi-dominant, including seeds and materials ofsaid cultivated plants and the progeny thereof. The present inventionalso discloses methods to produce Brassica oleracea cultivated plantsresistant to Albugo candida, methods to transfer the Albugo candidaresistance to susceptible or less resistant Brassica oleracea cultivatedplants. The present invention also further discloses molecular markerslinked to the resistance to Albugo candida.

In a 1^(st) embodiment, the invention relates to a cultivated Brassicaoleracea plant resistant to Albugo candida, comprising a resistancelocus, such locus being linked to a genetic determinant obtainable fromthe genome of a wild Brassica oleracea plant

In one embodiment, the invention relates to a cultivated Brassicaoleracea plant resistant to Albugo candida, wherein the geneticdeterminant linked to Albugo candida resistance locus is a qualitativeAlbugo candida resistance locus.

In one further embodiment, the plant according to the present inventionis a cultivated Brassica oleracea plant resistant to Albugo candidacomprising a resistance locus, wherein the resistance locus is locatedon chromosome 2.

In one further embodiment the invention relates to a cultivated Brassicaoleracea plant according to any of the preceding embodiments, whereinthe Albugo candida resistance locus is genetically linked to at leastone marker locus, which co-segregates with Albugo candida resistancetrait and comprises a marker that can be identified in a PCR reaction byamplification of a DNA fragment with a pair of PCR oligonucleotideprimers represented by a forward primer of SEQ ID No 1 and a reverseprimer of SEQ ID No 2 or any other marker located on chromosome 2 thatis statistically correlated and genetically linked to Albugo candidaresistance trait.

The present invention also concerns a cultivated Brassica oleracea plantaccording to any of the preceding embodiments, comprising at least oneallele or part thereof at a qualitative trait locus in the Brassicaoleracea genome contributing to resistance to Albugo candida, which isgenetically linked to at least one marker locus which co-segregates withAlbugo candida resistance trait and comprises a marker that can beidentified in a PCR reaction by amplification of a DNA fragment with apair of PCR oligonucleotide primers represented by a forward primer ofSEQ ID No 1 and a reverse primer of SEQ ID No 2, or any other markerlocated on chromosome 2 that is statistically correlated and geneticallylinked to Albugo candida resistance trait.

In one particular embodiment, the invention concerns a cultivatedBrassica oleracea plant according to previous embodiments whereinprimers pair amplifies a DNA fragment that comprises the at least onemarker locus which co-segregates with the Albugo candida resistancelocus.

More particularly the invention concerns cultivated Brassica oleraceaplant according to previous embodiment wherein primers pair amplifies aDNA fragment comprising at least one SNP within the at least one markerlocus which co-segregates with the Albugo candida resistance locus.

More particularly, the at least one SNP, within the at least one markerlocus which co-segregates with the Albugo candida resistance locus, isselected within the group comprising:

-   -   i. a SNP A represented by a T to C nucleotide exchange at        position 134 in the PCR amplified product    -   ii. a SNP B represented by a C to T nucleotide exchange at        position 108 in the PCR amplified product    -   ii. a SNP C represented by a T to C nucleotide exchange at        position 366 in the PCR amplified product.

In SNP A, C corresponds to resistant allele and T to susceptible allele.

In SNP B, T corresponds to resistant allele and C to susceptible allele.

In SNP C, C corresponds to resistant allele and T to susceptible allele.

The at least one SNP can be identified by sequencing the PCRamplification product to detect any nucleotide change. Nucleotidesequencing can be achieved by techniques known in the art such as Sangersequencing reaction followed by capillary electrophoresis.

The at least one SNP within the at least one marker locus whichco-segregates with the Albugo candida resistance locus can also beidentified by TaqMan allelic discrimination assay. Such TaqMan assay isan allelic discrimination PCR that uses fluorescent dye-labelled probesto determine which allele is present (Livak K J, Allelic discriminationusing fluorogenic probes and the 5′ nuclease assay. Gen Anal-Biomol Eng14: 143-149 (1999). When DNA from a susceptible plant is tested, theVIC-labelled S-selective probe anneals to its target sequence and isthen cleaved in the PCR by the 5′ nuclease activity of Taq DNApolymerase, thereby releasing the VIC dye away from its quencher andresulting in an increase in VIC fluorescence. Conversely, when DNA froma resistant plant is tested, only the FAM-labelled R-selective probeanneals, resulting in cleavage of this probe and an increase in FAMfluorescence. The relative fluorescence of these two dyes is measured inreal time during the PCR, with the endpoint values corrected forbackground and plotted against each other in a bivariate scatter plot.

In one particular embodiment, SNP A can be identified with a SNP A canbe identified with a pair of PCR oligonucleotide primers represented bya forward primer of SEQ ID No 3 and a reverse primer of SEQ ID No 4 andDNA probe of SEQ ID No 5 defining the resistant allele.

In one embodiment, the invention relates to a cultivated Brassicaoleracea plant according to any of the previous embodiments, comprisingat least one allele or part thereof at a qualitative trait locus in theBrassica oleracea genome contributing to resistance to Albugo candida,which is complementary to the corresponding allele present in Brassicaoleracea L var gemmifera UK 925, seed of which is deposited underDeposit Number NCIMB 41654, or in the progeny or in an ancestor thereof,and genetically linked to at least one marker locus in the genome ofBrassica oleracea L var gemmifera UK 925, seed of which is depositedunder deposit Number NCIMB 41654, or in the progeny or in an ancestorthereof, which marker locus co-segregates with the Albugo candidaresistance trait and comprises a marker that can be identified in a PCRreaction by amplification of a DNA fragment with a pair of PCRoligonucleotide primers represented by a forward primer of SEQ ID No 1and a reverse primer primer of SEQ ID No 2, or any other marker locatedon chromosome 2 that is statistically correlated and genetically linkedto Albugo candida resistance trait.

In one embodiment of the invention, a cultivated Brassica oleracea plantresistant to Albugo candida is provided, comprising a geneticdeterminant linked to Albugo candida resistance locus, wherein thegenetic determinant is obtainable from a donor plant having the geneticbackground of Brassica oleracea L var gemmifera UK 925, seed of which isdeposited under Deposit Number NCI MB 41654, or in the progeny or in anancestor thereof, comprising said genetic determinant or an Albugocandida resistance-conferring part thereof.

In one particular embodiment, a cultivated Brassica oleracea plantaccording to the present invention is provided, wherein the geneticdeterminant provides a monogenic and dominant, or at least semi-dominantresistance to Albugo candida.

In one embodiment, the present invention concerns a cultivated Brassicaoleracea plant, particularly a di-haploid, an inbred or a hybrid.

In yet another preferred embodiment, the cultivated plant according tothe present invention is male sterile, particularly cytoplasmic malesterile (CMS).

The male fertility of male sterile cultivated plants can be restored bymethods well-known in the art. The male fertility of CMS cultivatedplants, in particular CMS B. oleracea cultivated plants, is preferablyrestored by cell fusion. For this, cells of a CMS cultivated plant arefused to cells of a male fertile cultivated plant to replace the nucleusof the fertile cultivated plant by the nucleus of sterile cultivatedplant in the fertile cytoplasmic background, and restore fertility. Cellfusion techniques are well-known in the art and are for exampledescribed in Sigareva and Earle (1997) Theor. Appl. Genet. 94: 213-320.Using such techniques, male fertile cultivated plants are regenerated,and allowed to self-pollinate or crossed to another cultivated plant.

According to the present invention, seeds of cultivated Brassicaoleracea plant according to the different embodiments represented anddescribed herein comprising the genetic determinant contributing toresistance to Albugo candida, are provided.

In a particular embodiment, seed of cultivated Brassica oleracea plantaccording to the different embodiments represented and described hereinis hybrid seed.

Also, the present invention concerns seeds of cultivated Brassicaoleracea plant according to the different embodiments represented anddescribed herein wherein the resistance locus is located on chromosome2.

Brassica species and sub-species are for example described in P. H.Williams (Screening Crucifers for multiple disease resistance, Workshop1981, Un. Wisconsin-Madison) and have been further genetically analyzedin Song, K M et al. (TAG 75, 1988, 784-794; TAG 76, 1988, 593-600 andTAG 79, 1990, 497-506. Series of 3 articles).

In a preferred embodiment, Brassica oleracea cultivated plants of thepresent invention are for example: Brassica oleracea L. (cole-crops)var. acephala DC. (kales, collards) var. albiflora Sun [=B. alboglabra](Chinese kale) var. alboglabra [=B. alboglabra] (Chinese kale) var.botrytis L. (cauliflower, heading broccoli) var. capitata L. (cabbage)var. chinensis Prain (burma sarson) var. fimbriata Mill. (kitchen kale)var. fruticosa Metz. (thousand-head kale) var. gemmifera DC. (Brusselssprouts) var. gongylodes L. (kohlrabi) var. italica Plenck. (broccoli,calabrese) var. sabauda L. (savoy cabbage) var. tronchuda L. H. Bailey(tronchuda cabbage) var. costata (Portugese cabbage) var. medullosa(marrow stem kale) var. palmifolia (kale, Jersey kale) var. ramosa(thousand-head kale) var. sabellica (borecole). Preferred B. oleraceacultivated plants of the present invention are white cabbage,cauliflower, Brussels sprouts, Savoy cabbage and broccoli.

In one embodiment the present invention also provides plant materialfrom a cultivated Brassica oleracea plant according to any describedembodiment comprising leaves, stems, shoots, roots, flowers, flowerparts, buds, florets, pollen, egg cells, zygotes, seeds, cuttings, cellsor tissue cultures or any other part or product of the plant which stillexhibits the resistance phenotype, particularly when grown into a plant.

In one embodiment the cultivated Brassica oleracea plant according tothe present invention is a cultivated Brassica oleracea plant accordingto any of the preceding embodiments, which grows edible leaves, florets,buds, curds, stalk, and sprouts for human consumption.

In a further embodiment, the present invention relates to a method forproducing or selecting a cultivated Brassica oleracea plant, exhibitingresistance to Albugo candida, comprising the steps of:

-   -   a. selecting a plant of the genus Brassica, which exhibits        Albugo candida resistance, wherein said resistance is associated        with at least one genetic determinant or a functional part        thereof capable of directing or controlling expression of said        resistance to Albugo candida, wherein said genetic determinant        is genetically linked to at least one marker locus, which        co-segregates with the Albugo candida resistance trait and        comprises a marker that can be identified in a PCR reaction by        amplification of a DNA fragment with a pair of PCR        oligonucleotide primers represented by a forward primer of SEQ        ID No 1 and a reverse primer of SEQ ID No 2, or any other marker        located on chromosome 2 that is statistically correlated and        genetically linked to Albugo candida resistance trait;    -   b. crossing said plant of step a), which exhibits Albugo candida        resistance, with a Brassica plant, particularly a cultivated        Brassica oleracea plant, which is susceptible to Albugo candida        or exhibits a low level of resistance against Albugo candida,        and    -   c. selecting progeny from said cross which exhibits Albugo        candida resistance and demonstrates association with said at        least one marker locus of step a).

In a particular embodiment, in the method for producing or selecting acultivated Brassica oleracea plant resistant to Albugo candida asdescribed above, primer pair amplifies a DNA fragment comprising atleast one SNP within the at least one marker locus which co-segregateswith the Albugo candida resistance locus.

In a further particular embodiment the at least one SNP within the atleast one marker locus which segregates with the Albugo candidaresistance locus is selected within the group comprising:

-   -   i. a SNP A represented by a T to C nucleotide exchange at        position 134 in the PCR amplified product    -   ii. a SNP B represented by a C to T nucleotide exchange at        position 108 in the PCR amplified product    -   ii. a SNP C represented by a T to C nucleotide exchange at        position 366 in the PCR amplified product.

In SNP A, C corresponds to resistant allele and T to susceptible allele.

In SNP B, T corresponds to resistant allele and C to susceptible allele.

In SNP C, C corresponds to resistant allele and T to susceptible allele.

In an embodiment of the above selection or production method of acultivated Brassica oleracea plant resistant to Albugo candida, the SNPA can be identified with a pair of PCR oligonucleotide primersrepresented by a forward primer of SEQ ID No 3 and a reverse primer ofSEQ ID No 4 and a DNA probe of SEQ ID No 5 defining the resistantallele.

In a further embodiment, a method according to any one of the previousembodiments is provided for obtaining a cultivated Brassica oleraceaplant, resistant to Albugo candida, wherein the donor Brassica plant ofstep (a) is a Brassica oleracea plant according to any embodiments ofthe present invention, the method comprising the additional step ofbackcrossing the Albugo candida resistant Brassica oleracea plantobtained in step c) with the susceptible Brassica oleracea plant of stepb).

In one embodiment of the present invention, the determination of theassociation between Albugo candida resistance and the at least onemarker locus in step c) of the method here above described isaccomplished by carrying out a PCR reaction with the primers identifiedin step a).

The present invention also provides the use of cultivated Brassicaoleracea plants resistant to Albugo candida according to the differentembodiment of the present invention for the production of Brassicaoleracea plant part for human consumption.

In one particular embodiment, the use of cultivated Brassica oleraceaplants resistant to Albugo candida according to the previous embodimentcomprises plant part which is selected from the group comprising:leaves, bud, florets, curd, stem.

In a further embodiment, the invention provides a method for obtainingBrassica oleracea edible parts resistant to Albugo candida comprisingthe steps of

-   -   i. sowing a seed of plant according to the present invention        herein described or obtained in a method as herein claimed, and    -   ii. growing said plant in order to produce edible parts and        harvesting the said edible parts produced by said plant.

The present invention discloses the use of Albugo candida resistantpropagating material obtainable from a cultivated Brassica oleraceaplant according to the present invention herein described or obtained ina method as herein claimed for growing an Albugo candida resistantBrassica oleracea plant in order to produce edible parts and harvestsaid edible parts.

Edible parts of a plant according to the present invention compriseleaves, sprouts, stems, stalks, curds, florets and the like of a plantaccording to the different embodiment of the present invention.

Edible parts of a plant according to the present invention also compriseprocessed, including minimally processed, part of plant such as shreddedleaves, cut leaves, cut florets, cut sprouts, cut curds, cut stems andstalks.

Indeed beside the fact that Albugo candida induces abnormal growth anddecreased yield for production of cultivated Brassica species, it alsonegatively impacts the visual appearance of the edible part of theplant. The infection by the pathogen induces white or creamy pustulesfilled sporangia on leaves, stems, and floral parts of the plant. Allthese abnormalities constitute huge drawbacks regardingcommercialization of the edible part of the Brassica oleracea plant asdefinitively non-appealing regarding the consumer consideration.

Due to the resistance of the cultivated Brassica oleracea plantaccording to the present invention to Albugo candida, the edible part ofthe said plant have a much better appealing aspect for consumer. Suchappealing aspect is positive either for raw or fresh market or forintermediate and fully processed products. Such intermediate processedproducts may comprise, without limitation, shredded cabbage in bag,broccoli florets and cauliflower curds packed fresh or frozen, forexample.

The present invention also relates to the use of Albugo candidaresistant propagating material obtainable from a Brassica oleracea plantaccording to any of the preceding embodiments for growing an Albugocandida resistant plant in order to produce edible parts and harvestsaid edible parts.

In still another embodiment, the instant invention provides a method ofprotecting a field of Brassica oleracea plants, particularly Brassicaoleracea plants, against infection by Albugo candida, wherein saidmethod is characterized by planting a seed according to the presentinvention herein described or obtained in a method as herein claimed,and growing a cultivated Brassica oleracea plant which exhibits aresistance against Albugo candida. In a further particular embodiment,said Brassica plant or field is sprayed with a crop protection chemicalactive against Albugo candida at a lower concentration or lessfrequently than a Brassica oleracea plant not exhibiting saidresistance.

The present invention is particularly advantageous in that theresistance of the instant invention is easily transferred between B.oleracea cultivated plants and commercial lines. Higher yields areobtained because of the absence of disease on resistant cultivatedplants. Moreover much less crop protection chemicals or no cropprotection chemicals at all are required against Albugo candida when B.oleracea cultivated plants of the present invention are grown.

In one embodiment, the invention relates to a method for producinghybrid seeds of Brassica, particularly Brassica oleracea, resistant toAlbugo candida comprising the steps of:

-   -   i. planting a female, particularly a male sterile female plant,        and a male plant according to any of preceding embodiments        according to the present invention,    -   ii. effecting cross pollination between both parents,    -   iii. growing the plant till seed setting,    -   iv. collecting the seeds and    -   v. obtaining the hybrid seeds.

In a particular embodiment of the method of producing hybrid seedsaccording to the previous embodiment, the male sterile female parent isgenetic male sterile (GMS) or cytoplasmic male sterile (CMS).

In another preferred embodiment, the cultivated plant according to thepresent invention is homozygous or heterozygous for the resistance toAlbugo candida.

DEFINITIONS

The technical terms and expressions used within the scope of thisapplication are generally to be given the meaning commonly applied tothem in the pertinent art of plant breeding and cultivation if nototherwise indicated herein below.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a plant”includes one or more plants, and reference to “a cell” includes mixturesof cells, tissues, and the like.

A cultivated “Brassica” oleracea plant is understood within the scope ofthe invention to refer to a plant that is no longer in the natural statebut has been developed by human care and for human use and/orconsumption. “Cultivated Brassica oleracea plants” are furtherunderstood to exclude those wild-type species which comprise the traitbeing subject of this invention as a natural trait and/or part of theirnatural genetics.

A “genetic determinant contributing to resistance” is understood hereinto refer to a heritable genetic element that is capable of contributingto the resistance of the plant towards the pathogen by influencingexpression of this resistance trait on the level of the DNA itself, onthe level of translation, transcription and/or activation of a finalpolypeptide product, i.e., to down regulate and counter the infestationleading to the phenotypic expression of the resistance.

An “allele” is understood within the scope of the invention to refer toalternative or variant forms of various genetic units identical orassociated with different forms of a gene or of any kind of identifiablegenetic element, which are alternative in inheritance because they aresituated at the same locus in homologous chromosomes. Such alternativeor variant forms may be the result of single nucleotide polymorphisms,insertions, inversions, translocations or deletions, or the consequenceof gene regulation caused by, for example, by chemical or structuralmodification, transcription regulation or post-translationalmodification/regulation. In a diploid cell or organism, the two allelesof a given gene or genetic element typically occupy corresponding locion a pair of homologous chromosomes.

An allele associated with a qualitative trait may comprise alternativeor variant forms of various genetic units including those that areidentical or associated with a single gene or multiple genes or theirproducts or even a gene disrupting or controlled by a genetic factorcontributing to the phenotype represented by the locus.

As used herein, the term “marker allele” refers to an alternative orvariant form of a genetic unit as defined herein above, when used as amarker to locate genetic loci containing alleles on a chromosome thatcontribute to variability of phenotypic traits.

As used herein, the term “breeding”, and grammatical variants thereof,refer to any process that generates a progeny individual. Breedings canbe sexual or asexual, or any combination thereof. Exemplary non-limitingtypes of breedings include crossings, selfings, doubled haploidderivative generation, and combinations thereof.

As used herein, the phrase “established breeding population” refers to acollection of potential breeding partners produced by and/or used asparents in a breeding program; e.g., a commercial breeding program. Themembers of the established breeding population are typicallywell-characterized genetically and/or phenotypically. For example,several phenotypic traits of interest might have been evaluated, e.g.,under different environmental conditions, at multiple locations, and/orat different times. Alternatively or in addition, one or more geneticloci associated with expression of the phenotypic traits might have beenidentified and one or more of the members of the breeding populationmight have been genotyped with respect to the one or more genetic locias well as with respect to one or more genetic markers that areassociated with the one or more genetic loci.

As used herein, the phrase “diploid individual” refers to an individualthat has two sets of chromosomes, typically one from each of its twoparents. However, it is understood that in some embodiments a diploidindividual can receive its “maternal” and “paternal” sets of chromosomesfrom the same single organism, such as when a plant is selfed to producea subsequent generation of plants.

“Homozygous” is understood within the scope of the invention to refer tolike alleles at one or more corresponding loci on homologouschromosomes.

“Heterozygous” is understood within the scope of the invention to referto unlike alleles at one or more corresponding loci on homologouschromosomes.

“Backcrossing” is understood within the scope of the invention to referto a process in which a hybrid progeny is repeatedly crossed back to oneof the parents. Different recurrent parents may be used in subsequentbackcrosses.

“Locus” is understood within the scope of the invention to refer to aregion on a chromosome, which comprises a gene or any other geneticelement or factor contributing to a trait.

As used herein, “marker locus” refers to a region on a chromosome, whichcomprises a nucleotide or a polynucleotide sequence that is present inan individual's genome and that is associated with one or more loci ofinterest, which may which comprise a gene or any other genetic elementor factor contributing to a trait. “Marker locus” also refers to aregion on a chromosome, which comprises a polynucleotide sequencecomplementary to a genomic sequence, such as a sequence of a nucleicacid used as probes.

“Genetic linkage” is understood within the scope of the invention torefer to an association of characters in inheritance due to location ofgenes in proximity on the same chromosome, measured by percentrecombination between loci (centi-Morgan, cM).

For the purpose of the present invention, the term “co-segregation”refers to the fact that the allele for the trait and the allele(s) forthe marker(s) tend to be transmitted together because they arephysically close together on the same chromosome (reduced recombinationbetween them because of their physical proximity) resulting in anon-random association of their alleles as a result of their proximityon the same chromosome. “Co-segregation” also refers to the presence oftwo or more traits within a single plant of which at least one is knownto be genetic and which cannot be readily explained by chance.

As used herein, the term “genetic architecture at the qualitative traitlocus” refers to a genomic region which is statistically correlated tothe phenotypic trait of interest and represents the underlying geneticbasis of the phenotypic trait of interest.

As used herein, the phrases “sexually crossed” and “sexual reproduction”in the context of the presently disclosed subject matter refers to thefusion of gametes to produce progeny (e.g., by fertilization, such as toproduce seed by pollination in plants). A “sexual cross” or“cross-fertilization” is in some embodiments fertilization of oneindividual by another (e.g., cross-pollination in plants). The term“selfing” refers in some embodiments to the production of seed byself-fertilization or self-pollination; i.e., pollen and ovule are fromthe same plant.

As used herein, the phrase “genetic marker” refers to a feature of anindividual's genome (e.g., a nucleotide or a polynucleotide sequencethat is present in an individual's genome) that is associated with oneor more loci of interest. In some embodiments, a genetic marker ispolymorphic in a population of interest, or the locus occupied by thepolymorphism, depending on context. Genetic markers include, forexample, single nucleotide polymorphisms (SNPs), indels (i.e.,insertions/deletions), simple sequence repeats (SSRs), restrictionfragment length polymorphisms (RFLPs), random amplified polymorphic DNAs(RAPDs), cleaved amplified polymorphic sequence (CAPS) markers,Diversity Arrays Technology (DArT) markers, and amplified fragmentlength polymorphisms (AFLPs), among many other examples. Genetic markerscan, for example, be used to locate genetic loci containing alleles on achromosome that contribute to variability of phenotypic traits. Thephrase “genetic marker” can also refer to a polynucleotide sequencecomplementary to a genomic sequence, such as a sequence of a nucleicacid used as probes.

A genetic marker can be physically located in a position on a chromosomethat is within or outside of the genetic locus with which it isassociated (i.e., is intragenic or extragenic, respectively). Statedanother way, whereas genetic markers are typically employed when thelocation on a chromosome of the gene or of a functional mutation, e.g.within a control element outside of a gene, that corresponds to thelocus of interest has not been identified and there is a non-zero rateof recombination between the genetic marker and the locus of interest,the presently disclosed subject matter can also employ genetic markersthat are physically within the boundaries of a genetic locus (e.g.,inside a genomic sequence that corresponds to a gene such as, but notlimited to a polymorphism within an intron or an exon of a gene). Insome embodiments of the presently disclosed subject matter, the one ormore genetic markers comprise between one and ten markers, and in someembodiments the one or more genetic markers comprise more than tengenetic markers.

As used herein, the term “genotype” refers to the genetic constitutionof a cell or organism. An individual's “genotype for a set of geneticmarkers” includes the specific alleles, for one or more genetic markerloci, present in the individual's haplotype. As is known in the art, agenotype can relate to a single locus or to multiple loci, whether theloci are related or unrelated and/or are linked or unlinked. In someembodiments, an individual's genotype relates to one or more genes thatare related in that the one or more of the genes are involved in theexpression of a phenotype of interest. Thus, in some embodiments agenotype comprises a summary of one or more alleles present within anindividual at one or more genetic loci of a quantitative trait. In someembodiments, a genotype is expressed in terms of a haplotype (definedherein below).

As used herein, the term “germplasm” refers to the totality of thegenotypes of a population or other group of individuals (e.g., aspecies). The term “germplasm” can also refer to plant material; e.g., agroup of plants that act as a repository for various alleles. The phrase“adapted germplasm” refers to plant materials of proven geneticsuperiority; e.g., for a given environment or geographical area, whilethe phrases “non-adapted germplasm,” “raw germplasm,” and “exoticgermplasm” refer to plant materials of unknown or unproven geneticvalue; e.g., for a given environment or geographical area; as such, thephrase “non-adapted germplasm” refers in some embodiments to plantmaterials that are not part of an established breeding population andthat do not have a known relationship to a member of the establishedbreeding population.

As used herein, the terms “hybrid”, “hybrid plant,” and “hybrid progeny”refers to an individual produced from genetically different parents(e.g., a genetically heterozygous or mostly heterozygous individual).

As used herein, the phrase “single cross F₁ hybrid” refers to an F₁hybrid produced from a cross between two inbred lines.

As used herein, the phrase “inbred line” refers to a geneticallyhomozygous or nearly homozygous population. An inbred line, for example,can be derived through several cycles of brother/sister breedings or ofselfing or in dihaploid production. In some embodiments, inbred linesbreed true for one or more phenotypic traits of interest. An “inbred”,“inbred individual” or “inbred progeny” is an individual sampled from aninbred line.

As used herein, the term “dihaploid line”, refers to stable inbred linesissued from another culture. Some pollen grains (haploid) cultivated onspecific medium and circumstances can develop plantlets containing nchromosomes. These plantlets are then “doubled” and contain 2nchromosomes. The progeny of these plantlets are named “dihaploid” andare essentially not segregating any more (stable).

As used herein, the term “linkage”, and grammatical variants thereof,refers to the tendency of alleles at different loci on the samechromosome to segregate together more often than would be expected bychance if their transmission were independent, in some embodiments as aconsequence of their physical proximity.

As used herein, the phrase “nucleic acid” refers to any physical stringof monomer units that can be corresponded to a string of nucleotides,including a polymer of nucleotides (e.g., a typical DNA, cDNA or RNApolymer), modified oligonucleotides (e.g., oligonucleotides comprisingbases that are not typical to biological RNA or DNA, such as2′-O-methylated oligonucleotides), and the like. In some embodiments, anucleic acid can be single-stranded, double-stranded, multi-stranded, orcombinations thereof. Unless otherwise indicated, a particular nucleicacid sequence of the presently disclosed subject matter optionallycomprises or encodes complementary sequences, in addition to anysequence explicitly indicated.

As used herein, the phrase “phenotypic trait” refers to the appearanceor other detectable characteristic of an individual, resulting from theinteraction of its genome, proteome and/or metabolome with theenvironment.

As used herein, the phrase “resistance” refers to the ability of a plantto restrict the growth and development of a specified pathogen and/orthe damage they cause when compared to susceptible plants under similarenvironmental conditions and pathogen pressure. Resistant plants mayexhibit some disease symptoms or damage under pathogen pressure, e.g.fungus.

According to European Seed Association and as used therein, the term“resistance” includes “standard resistance” and “intermediateresistance”

“Standard resistance” refers to plants that highly restrict the growthand development of the specified pest or pathogen under normal pest orpathogen pressure and/or are sufficiently unattractive to the specifiedpest or pathogen so that they exhibit no or only minor disease symptomsor damage when compared to susceptible counterparts. These plants may,however, exhibit some disease symptoms or damage under heavy pest orpathogen pressure.

“Intermediate resistance” refers to plants that distract insects and/orrestrict the growth and development of the specified pest or pathogen,or show reduced damage compared to susceptible counterparts but mayexhibit a greater range of symptoms or damage compared to standardresistant plants. Intermediate resistant plants will still showsignificantly less severe symptoms or damage than susceptible plantswhen grown under similar environmental conditions and/or pest orpathogen pressure

As used herein, the phrase “susceptibility” refers to the inability of aplant to adequately restrict the growth and development of a specifiedpathogen.

As used herein, the phrase “Albugo resistance” or “resistance to Albugocandida” or “Albugo resistant plant” refers to the plants capability toresist colonization by the fungus. Albugo resistance is determinedwithin the scope of the present invention in a pathotest as described indetail in Example 1. Particularly a plant resistant to Albugo candida inthe context of the present invention shows a score comprised between 6and 9 in the scale according to pathotest of Example 1.

As used herein, the term “plurality” refers to more than one. Thus, a“plurality of individuals” refers to at least two individuals. In someembodiments, the term plurality refers to more than half of the whole.For example, in some embodiments a “plurality of a population” refers tomore than half the members of that population.

As used herein, the term “progeny” refers to the descendant(s) of aparticular cross. Typically, progeny result from breeding of twoindividuals, although some species (particularly some plants andhermaphroditic animals) can be selfed (i.e., the same plant acts as thedonor of both male and female gametes). The descendant(s) can be, forexample, of the F₁, the F₂, or any subsequent generation.

As used herein, the phrase “qualitative trait” refers to a phenotypictrait that is controlled by one or a few genes that exhibit majorphenotypic effects. Because of this, qualitative traits are typicallysimply inherited. Examples in plants include, but are not limited to,flower color, fruit color, and several known disease resistances suchas, for example, Fungus spot resistance.

“Marker-based selection” is understood within the scope of the inventionto refer to e.g. the use of genetic markers to detect one or morenucleic acids from the plant, where the nucleic acid is associated witha desired trait to identify plants that carry genes for desirable (orundesirable) traits, so that those plants can be used (or avoided) in aselective breeding program.

“Microsatellite or SSRs (Simple Sequence Repeats) Marker” is understoodwithin the scope of the invention to refer to a type of genetic markerthat consists of numerous repeats of short sequences of DNA bases, whichare found at loci throughout the plant's genome and have a likelihood ofbeing highly polymorphic.

“PCR (Polymerase chain reaction)” is understood within the scope of theinvention to refer to a method of producing relatively large amounts ofspecific regions of DNA or subset(s) of the genome, thereby makingpossible various analyses that are based on those regions.

“PCR primer” is understood within the scope of the invention to refer torelatively short fragments of single-stranded DNA used in the PCRamplification of specific regions of DNA.

“Phenotype” is understood within the scope of the invention to refer toa distinguishable characteristic(s) of a genetically controlled trait.

As used therein “trait” refers to characteristic or phenotype, forexample a resistance to a disease. A trait may be inherited in adominant or recessive manner, or may be monogenic or polygenic. A traitis for example a resistance to a disease.

As used herein, the phrase “phenotypic trait” refers to the appearanceor other detectable characteristic of an individual, resulting from theinteraction of its genome, proteome and/or metabolome with theenvironment.

“Polymorphism” is understood within the scope of the invention to referto the presence in a population of two or more different forms of agene, genetic marker, or inherited trait or a gene product obtainable,for example, through alternative splicing, DNA methylation, etc.

“Selective breeding” is understood within the scope of the invention torefer to a program of breeding that uses plants that possess or displaydesirable traits as parents.

“Tester” plant is understood within the scope of the invention to referto a plant of the genus Brassica used to characterize genetically atrait in a plant to be tested. Typically, the plant to be tested iscrossed with a “tester” plant and the segregation ratio of the trait inthe progeny of the cross is scored.

“Probe” as used herein refers to a group of atoms or molecules which iscapable of recognising and binding to a specific target molecule orcellular structure and thus allowing detection of the target molecule orstructure. Particularly, “probe” refers to a labeled DNA or RNA sequencewhich can be used to detect the presence of and to quantitate acomplementary sequence by molecular hybridization.

The term “hybridize” as used herein refers to conventional hybridizationconditions, preferably to hybridization conditions at which 5×SSPE, 1%SDS, 1×Denhardts solution is used as a solution and/or hybridizationtemperatures are between 35° C. and 70° C., preferably 65° C. Afterhybridization, washing is preferably carried out first with 2×SSC, 1%SDS and subsequently with 0.2×SSC at temperatures between 35° C. and 75°C., particularly between 45° C. and 65° C., but especially at 59° C.(regarding the definition of SSPE, SSC and Denhardts solution seeSambrook et al. loc. cit.). High stringency hybridization conditions asfor instance described in Sambrook et al, supra, are particularlypreferred. Particularly preferred stringent hybridization conditions arefor instance present if hybridization and washing occur at 65° C. asindicated above. Non-stringent hybridization conditions for instancewith hybridization and washing carried out at 45° C. are less preferredand at 35° C. even less.

“Sequence Homology or Sequence Identity” is used herein interchangeably.The terms “identical” or percent “identity” in the context of two ormore nucleic acid or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection. If twosequences which are to be compared with each other differ in length,sequence identity preferably relates to the percentage of the nucleotideresidues of the shorter sequence which are identical with the nucleotideresidues of the longer sequence. Sequence identity can be determinedconventionally with the use of computer programs such as the Bestfitprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, 575 Science DriveMadison, Wis. 53711). Bestfit utilizes the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2 (1981), 482-489,in order to find the segment having the highest sequence identitybetween two sequences. When using Bestfit or another sequence alignmentprogram to determine whether a particular sequence has for instance 95%identity with a reference sequence of the present invention, theparameters are preferably so adjusted that the percentage of identity iscalculated over the entire length of the reference sequence and thathomology gaps of up to 5% of the total number of the nucleotides in thereference sequence are permitted. When using Bestfit, the so-calledoptional parameters are preferably left at their preset (“default”)values. The deviations appearing in the comparison between a givensequence and the above-described sequences of the invention may becaused for instance by addition, deletion, substitution, insertion orrecombination. Such a sequence comparison can preferably also be carriedout with the program “fasta20u66” (version 2.0u66, September 1998 byWilliam R. Pearson and the University of Virginia; see also W. R.Pearson (1990), Methods in Enzymology 183, 63-98, appended examples andhttp://workbench.sdsc.edu/). For this purpose, the “default” parametersettings may be used.

Another indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other understringent conditions. The phrase: “hybridizing specifically to” refersto the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point forthe specific sequence at a defined ionic strength and pH. Typically,under “stringent conditions” a probe will hybridize to its targetsubsequence, but to no other sequences.

The thermal melting point is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Very stringent conditions are selected to beequal to the T.sub.m for a particular probe. An example of stringenthybridization conditions for hybridization of complementary nucleicacids which have more than 100 complementary residues on a filter in aSouthern or northern blot is 50% formamide with 1 mg of heparin at 42°C., with the hybridization being carried out overnight. An example ofhighly stringent wash conditions is 0.1 5M NaCl at 72° C. for about 15minutes. An example of stringent wash conditions is a 0.2 times SSC washat 65° C. for 15 minutes (see, Sambrook, infra, for a description of SSCbuffer). Often, a high stringency wash is preceded by a low stringencywash to remove background probe signal. An example medium stringencywash for a duplex of, e.g., more than 100 nucleotides, is 1 times SSC at45° C. for 15 minutes. An example low stringency wash for a duplex of,e.g., more than 100 nucleotides, is 4-6 times SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.0MNa ion, typically about 0.01 to 1.0 M Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2 times (or higher) than that observed for an unrelated probein the particular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g. when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

A “plant” is any plant at any stage of development, particularly a seedplant.

A “plant cell” is a structural and physiological unit of a plant,comprising a protoplast and a cell wall. The plant cell may be in formof an isolated single cell or a cultured cell, or as a part of higherorganized unit such as, for example, plant tissue, a plant organ, or awhole plant.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment.

“Plant material” or “plant material obtainable from a plant” refers toleaves, stems, roots, flowers or flower parts, fruits, pollen, eggcells, zygotes, seeds, cuttings, cell or tissue cultures, or any otherpart or product of a plant.

A “plant organ” is a distinct and visibly structured and differentiatedpart of a plant such as a root, stem, leaf, flower bud, or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

The terms “race” or “races” refer to any inbreeding group, includingtaxonomic subgroups such as subspecies, taxonomically subordinate to aspecies and superordinate to a subrace and marked by a pre-determinedprofile of latent factors of hereditary traits.

As used herein, the term “population” means a genetically heterogeneouscollection of plants sharing a common genetic derivation

As used herein, the term “variety” or “cultivar” means a group ofsimilar plants that by structural features and performance can beidentified from other varieties within the same species.

As used therein “semi dominance” means incomplete dominance; theproduction of an intermediate phenotype in individuals heterozygous forthe gene concerned; it is generally considered to be a type ofincomplete dominance, with the heterozygote resembling one homozygotemore than the other.

As used therein “dominant” means: a gene that produces the samephenotypic character when its alleles are present in a single dose(heterozygous) per nucleus, as it does in a double dose (homozygous).

As used therein “recessive” means for a gene and/or allele whosephenotypic effect is expressed in the homozygous state but masked in thepresence of the dominant allele in the heterozygous state.

In one embodiment, the present invention relates to novel Albugo candidaresistant Brassica plants, particularly Brassica oleracea plants, andlines, and improved methods for producing them utilizing the molecularmarkers described herein in selective breeding techniques. Brassicaplants that do not contain at least one of the marker locus identifiedherein are susceptible to infection by Albugo candida.

In particular, the at least one marker locus controlling the Albugocandida resistance is located within genome of Albugo candida resistantBrassica oleracea plant. Molecular markers co-segregating with theAlbugo candida resistance can be identified using marker-assistedselection, the techniques for which are well known in the art. Markersthat can be used in such selection techniques are represented by atleast one oligonucleotide primer selected from the group of primersgiven in SEQ ID NO:1, SEQ ID NO: 2, by any adjacent marker that isstatistically correlated and thus co-segregates with the Albugo candidaresistance trait.

In one aspect, the plant according to the present invention may beobtained by introgressing the Albugo candida resistance trait from anancestor plant, particularly a wild ancestor plant into a cultivatedBrassica plant, particularly a cultivated Brassica oleracea plant.

In one specific embodiment, of the invention, the wild ancestor fromwhich Albugo candida resistance trait may be obtained is a wild typeBrassica oleracea, particularly wild primitive Brassica oleraceaacephala HRI 1205, or from an progeny or an ancestor thereof comprisingsaid resistance trait locus.

For this accession the following source history can be provided:

-   -   Accession was collected in Portugal in 4 Oct. 1993.    -   Locality: Quinta da Igreja. Macainhas de baixo guarda.    -   Donors: UNKFARIAS ASTLEY & CHEUNG SO MUI.    -   Maintained by Warwick HRI Genetic Resources Unit University of        Warwick, Wellesbourne, Warwick, UK.        The resistance trait according to the present invention, which        confers to a plant expressing this trait, resistance to        infestations with Albugo candida, may in the alternative, be        obtained from Brassica oleracea L var gemmifera UK 925, seed of        which is deposited under deposit Number NCIMB 41654.

Brassica oleracea L var gemmifera UK 925 resulted from a cross ofBrassica oleracea acephala HRI 1205, as the donor of the resistancetrait with a Brussels sprout inbred line. Albugo resistant progeny ofthis cross was further back-crossed with the recipient Brussels sproutline and then selfed several times in order to have a fixed line forresistance. The fixed line for resistance was then crossed with a femaleline holding CMS in order to obtain the UK 925 Brassica oleracea L vargemmifera, seed of which is deposited under deposit Number NCIMB 41654.

Moreover, other accessions of related Brassica oleracea species can beexamined for the presence of at least one of the marker locus identifiedherein by using the markers of the present invention.

The molecular markers provided herein and co-segregating with at leastone marker locus contributing to Albugo candida resistance, may be usedto introgress one or more of said marker locus from a first donor plantinto a second recipient plant.

Based on the description of the present invention, the skilled personwho is in possession of Brassica oleracea L var gemmifera UK 925 or ofan ancestor containing a trait locus associated with Albugo candidaresistance, has no difficulty to transfer the Albugo candida resistancetrait of the present invention to other cultivated Brassica oleraceaplants of various type and cultivars using breeding techniques wellknown in the art.

Thus the trait of the present invention can be transferred to cultivatedBrassica oleracea plant such as Brassica oleracea L. (cole-crops) var.acephala DC. (kales, collards) var. albiflora Sun [=B. alboglabra](Chinese kale) var. alboglabra [=B. alboglabra] (Chinese kale) var.botrytis L. (cauliflower, heading broccoli) var. capitata L. (cabbage)var. chinensis Prain (burma sarson) var. fimbriata Mill. (kitchen kale)var. fruticosa Metz. (thousand-head kale) var. gemmifera DC. (Brusselssprouts) var. gongylodes L. (kohlrabi) var. italica Plenck. (broccoli,calabrese) var. sabauda L. (savoy cabbage) var. tronchuda L. H. Bailey(tronchuda cabbage) var. costata (Portugese cabbage) var. medullosa(marrow stem kale) var. palmifolia (kale, Jersey kale) var. ramosa(thousand-head kale) var. sabellica (borecole).

The recipient plant is preferably a cultivated Brassica plant,particularly a cultivated Brassica oleracea, more particularly acultivated Brassica oleracea selected from the group comprising:Brassica oleracea L. (cole-crops) var. acephala DC. (kales, collards)var. albiflora Sun [=B. alboglabra] (Chinese kale) var. alboglabra [=B.alboglabra] (Chinese kale) var. botrytis L. (cauliflower, headingbroccoli) var. capitata L. (cabbage) var. chinensis Prain (burma sarson)var. fimbriata Mill. (kitchen kale) var. fruticosa Metz. (thousand-headkale) var. gemmifera DC. (Brussels sprouts) var. gongylodes L.(kohlrabi) var. italica Plenck. (broccoli, calabrese) var. sabauda L.(savoy cabbage) var. tronchuda L. H. Bailey (tronchuda cabbage) var.costata (Portugese cabbage) var. medullosa (marrow stem kale) var.palmifolia (kale, Jersey kale) var. ramosa (thousand-head kale) var.sabellica (borecole). Preferred recipient Brassica oleracea cultivatedplants of the present invention are white cabbage, cauliflower, Brusselssprouts, savoy cabbage and broccoli

Cultivated Brassica plants developed according to the present inventioncan advantageously derive a majority of their traits from the recipientplant, and derive Albugo candida resistance from the first donor plant.According to one aspect of the present invention, genes that encode forAlbugo candida resistance are mapped by identifying molecular markerslinked to resistance qualitative trait loci, the mapping utilizing a mixof resistant and susceptible to Albugo candida inbred Brassica plantsfor phenotypic scoring. Molecular characterization of such lines can beconducted using the techniques described by Monforte and Tanksley inGenome, 43: 803-813 (2000).

In another embodiment of the present invention, the present inventionrelates to methods for producing superior new Albugo candida resistantBrassica plants. In the method of the present invention, one or moregenes encoding for Albugo candida resistance are introgressed from adonor parental plant that is resistant to Albugo candida into arecipient cultivated Brassica plant that is either non-resistant or aplant that has a low level of resistance to infection by Albugo candida.The Albugo candida resistant cultivated Brassica oleracea plantsaccording to the present embodiments of the invention or producedaccording to the methods of the present invention can be either inbred,hybrid, or dihaploid Brassica plants.

The introgression of one or more genes encoding for Albugo candidaresistance into a recipient Brassica plant that is non-resistant orpossesses a low level of resistance to Albugo candida can beaccomplished using techniques known in the art. For example, one or moregenes encoding for Albugo candida resistance can be introgressed into arecipient Brassica oleracea plant that is non-resistant or a plant thathas a low level of resistance to Albugo candida using traditionalbreeding techniques.

The resistance to Albugo candida was transferred to different species ofcultivated Brassica oleraceas, in particular white cabbage, cauliflower,broccoli and Brussels sprouts, using standard breeding techniqueswell-known in the Brassica art. The trait was also further introgressedinto cultivated Brassica oleracea elite lines. The introgression of theresistance into the elite line can for example be achieved by recurrentselection breeding, for example by backcrossing.

In this case, the elite line (recurrent parent) is first crossed to adonor inbred (the non-recurrent parent) that carries the resistance. Theprogeny of this cross is then crossed back to the recurrent parentfollowed by selection in the resultant progeny for Albugo candidaresistance. After two, preferably three, preferably four, morepreferably five or more generations of backcrosses with the recurrentparent with selection for Albugo candida resistance, the progeny isheterozygous for the locus harbouring the resistance, but is like therecurrent parent for most or almost all other genes (see, for example,Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr(1987) Principles of Cultivar Development, Vol. 1: Theory and Technique,360-376, incorporated herein by reference). Selection for Albugo candidaresistance is carried out after each cross.

The cultivated Brassica oleracea plants according to the presentinvention and as described herein can be used in commercial Brassicaoleracea seeds production. Commercial Brassica oleracea plants aregenerally hybrids produced from the cross of two parental lines(inbreds). The development of hybrids requires, in general, thedevelopment of homozygous inbred lines, the crossing of these lines, andthe evaluation of the crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Breeding programscombine the genetic backgrounds from two or more inbred lines or variousother germplasm sources into breeding pools from which new inbred linesare developed by selfing and selection of desired phenotypes andcharacteristics. The new inbreds are crossed with other inbred lines andthe hybrids from these crosses are evaluated to determine which of thosehave commercial potential. Plant breeding and hybrid development areexpensive and labour and time-consuming processes. Pedigree breedingstarts with the crossing of two genotypes, each of which may have one ormore commercially desirable characteristics, such as, but not limitedto, disease resistance, insect resistance, valuable fruitcharacteristics, increased yield, etc. that is lacking in the other orwhich complements the other. If the two original parents do not provideall the desired characteristics, other sources can be included in thebreeding population in order to generate an established breedingpopulation. In the pedigree method, superior plants are selfed andselected in successive generations. In the succeeding generations theheterozygous condition gives way to homogeneous lines as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding five or more generations of selfing and selection is practiced:F1 to F2; F3 to F4; F4 to F5, etc. A single cross hybrid results fromthe cross of two inbred lines, each of which has a genotype thatcomplements the genotype of the other. The hybrid progeny of the firstgeneration is designated F1. In the development of commercial hybridsonly the F1 hybrid plants are sought. Preferred F1 hybrids are morevigorous than their inbred parents. This hybrid performance (hybridvigor or heterosis), can be manifested in many polygenic traits,including increased vegetative growth and increased yield. Brassicaplants can be easily cross-pollinated. A trait is also readilytransferred from one Brassica plant to another plant, including Brassicaplants of different types using conventional breeding techniques, forexample to further obtain commercial lines. The introgression of a traitinto the elite line is for example achieved by recurrent selectionbreeding, for example by backcrossing. In this case, the elite line(recurrent parent) is first crossed to a donor inbred (the non-recurrentparent) that carries the trait, particularly the “Albugo candidaresistance” trait according to the present invention. The progeny ofthis cross is then mated back to the recurrent parent followed byselection in the resultant progeny for the trait. After three,preferably four, more preferably five or more generations of backcrosseswith the recurrent parent with selection for the trait, particularly the“Albugo candida resistance” trait according to the present invention,the progeny is heterozygous for the locus harbouring the resistance, butis like the recurrent parent for most or almost all other genes (see,for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed.,172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1: Theoryand Technique, 360-376, incorporated herein by reference). Selection forthe trait is carried out after each cross. Male sterility is availablein Brassica. In particular cytoplasmic male sterility may be used incommercial lines e.g. Brassica oleracea cauliflower lines.

The population can be screened in a number of different ways. First, thepopulation can be screened using a traditional pathology disease screen.Such pathology disease screens are known in the art. Specifically, theindividual plants or parts thereof can be challenged in an incubator orgreenhouse with Albugo candida and the resulting resistant orsusceptible phenotypes of each plant scored. By way of example, and notof limitation, plants can be screened in a greenhouse as follows.

First, Brassica seeds are planted and grown to seedlings (approximatetime is 3-4 weeks) in a greenhouse—with preferably >10 plants per lineare evaluated. Since Resistance is not fully expressed in thecotyledons, therefore true leaves (preferably 3-4 leaf stage) are usedto inoculate plants The leaves can then be rated separately using adisease rating scale of 1-9 (1 to 5=susceptible and 6 to 9=resistantwith sub levels of intermediate resistance (6-7) and standard resistance(8-9)). The scale of the disease rating depends upon the presence andthe size of Albugo candida pustules on the leaves.

Albugo candida spores can be collected with a vacuum pump from ripenedpustules on affected material and dry stored at −20° C. (Gilijamse etal., 2004). Sporangia were suspended in cold demi-water and stored for 2hr at 5° C. to allow the sporangia to germinate into zoospores. Thezoospores can then be sprayed onto the test plants (concentration10⁻⁴-10⁻⁵ zoospores/ml) in a climate chamber with 100% RH. Incubationduring 10-14 days at 18-20° C. in a greenhouse after which the whitepustules start to appear. Final observation is done using a 1-9 scale inwhich 1=plants are fully covered with large pustules, and, 9=plants arehealthy without showing any symptoms. Plants scoring 1-5 on this scaleare regarded as being susceptible and plants scoring 6-9 are classifiedas being resistant; 6-7 is regarded as intermediately resistant and 8-9as standard resistant.

Second, marker-assisted selection can be performed using one or more ofthe hereinbefore described molecular markers to identify those hybridplants that contain one or more of the genes that encode for Albugocandida resistance. Alternatively, marker assisted selection can be usedto confirm the results obtained from the pathology screen. F2 hybridplants exhibiting a Albugo candida resistant phenotype contain therequisite genes encoding for Albugo resistance, and possess commerciallydesirable characteristics, are then selected and selfed for a number ofgenerations in order to allow for the Brassica plant to becomeincreasingly inbred. This process of continued selfing and selection canbe performed for five or more generations. The result of such breedingand selection is the production of lines that are genetically homogenousfor the genes associated with Albugo candida resistance as well as othergenes associated with traits of commercial interest. Alternatively, anew and superior Albugo candida resistant inbred Brassica oleracea plantline can be developed using the techniques of recurrent selection andbackcrossing. In this method, Albugo candida resistance can beintrogressed into a target recipient plant (which is called therecurrent parent) by crossing the recurrent parent with a first donorplant (which is different from the recurrent parent and referred toherein as the “non-recurrent parent”). The recurrent parent is a plantthat is non-resistant or has a low level of resistance to Albugo candidaand possesses commercially desirable characteristics, such as, but notlimited to disease resistance, insect resistance, valuable agronomiccharacteristics, etc.

The non-recurrent parent exhibits Albugo candida resistance and containsone or more genes that encode for Albugo candida resistance. Thenon-recurrent parent can be any plant variety or inbred line that iscross-fertile with the recurrent parent. The progeny resulting from across between the recurrent parent and non-recurrent parent arebackcrossed to the recurrent parent. The resulting plant population isthen screened. The population can be screened in a number of differentways. First, the population can be screened using a traditionalpathology screen as described previously herein, particularly inEXAMPLE 1. Second, marker-assisted selection can be performed using oneor more of the hereinbefore described molecular markers to identifythose progeny that contain one or more of genes encoding for Albugocandida resistance. Alternatively, marker-assisted selection can be usedto confirm the results obtained from the pathology screen. Once theappropriate selections are made, the process is repeated. The process ofbackcrossing to the recurrent parent and selecting for Albugo candidaresistance is repeated for approximately five or more generations. Theprogeny resulting from this process are heterozygous for one or moregenes that encode for Albugo candida resistance. The last backcrossgeneration is then selfed in order to provide for homozygous purebreeding progeny for Albugo candida resistance. The Albugo candidaresistant inbred Brassica oleracea lines described herein can be used inadditional crossings to create Albugo candida resistant hybrid plants.For example, a first Albugo candida resistant inbred Brassica oleraceaplant can be crossed with a second inbred Brassica oleracea plantpossessing commercially desirable traits such as, but not limited to,disease resistance, insect resistance, desirable agronomiccharacteristics, etc. This second inbred Brassica line may or may not beresistant to Albugo candida. The marker-assisted selection used in thehereinbefore described methods can be made, for example, step-wise,whereby the differenti Albugo candida resistant genes are selected inmore than one generation; or, as an alternative example, simultaneously,whereby all resistance genes are selected in the same generation.Marker-assisted selection for Albugo candida resistance may be donebefore, in conjunction with, or after testing and selection for othercommercially desirable input or output traits.

There are several types of molecular markers that may be used inmarker-based selection including, but not limited to, restrictionfragment length polymorphism (RFLP), random amplification of polymorphicDNA (RAPD), amplified restriction fragment length polymorphism (AFLP),single sequence repeats (SSR) and single nucleotide polymorphisms SNPs.

RFLP involves the use of restriction enzymes to cut chromosomal DNA atspecific short restriction sites, polymorphisms result from duplicationsor deletions between the sites or mutations at the restriction sites.

RAPD utilizes low stringency polymerase chain reaction (PCR)amplification with single primers of arbitrary sequence to generatestrain-specific arrays of anonymous DNA fragments. The method requiresonly tiny DNA samples and analyses a large number of polymorphic loci.

AFLP requires digestion of cellular DNA with a restriction enzyme(s)before using PCR and selective nucleotides in the primers to amplifyspecific fragments. With this method, using electrophoresis techniquesto visualize the obtained fragments, up to 100 polymorphic loci can bemeasured per primer combination and only small DNA sample are requiredfor each test.

SSR analysis is based on DNA micro-satellites (short-repeat) sequencesthat are widely dispersed throughout the genome of eukaryotes, which areselectively amplified to detect variations in simple sequence repeats.Only tiny DNA samples are required for an SSR analysis. SNPs use PCRextension assays that efficiently pick up point mutations. The procedurerequires little DNA per sample. One or two of the above methods may beused in a typical marker-based selection breeding program.

The most preferred method of achieving amplification of nucleotidefragments that span a polymorphic region of the plant genome employs thepolymerase chain reaction (“PCR”) (Mullis et al., Cold Spring HarborSymp. Quant. Biol. 51:263 273 (1986)), using primer pairs involving aforward primer and a backward primer that are capable of hybridizing tothe proximal sequences that define a polymorphism in its double-strandedform.

Alternative methods may be employed to amplify fragments, such as the“Ligase Chain Reaction” (“LCR”) (Barany, Proc. Natl. Acad. Sci. (U.S.A.)88:189 193 (1991)), which uses two pairs of oligonucleotide probes toexponentially amplify a specific target. The sequences of each pair ofoligonucleotides are selected to permit the pair to hybridize toabutting sequences of the same strand of the target. Such hybridizationforms a substrate for a template-dependent ligase. As with PCR, theresulting products thus serve as a template in subsequent cycles and anexponential amplification of the desired sequence is obtained.

LCR can be performed with oligonucleotides having the proximal anddistal sequences of the same strand of a polymorphic site. In oneembodiment, either oligonucleotide will be designed to include theactual polymorphic site of the polymorphism. In such an embodiment, thereaction conditions are selected such that the oligonucleotides can beligated together only if the target molecule either contains or lacksthe specific nucleotide that is complementary to the polymorphic sitepresent on the oligonucleotide. Alternatively, the oligonucleotides maybe selected such that they do not include the polymorphic site (see,Segev, PCT Application WO 90/01069).

A further method that may alternatively be employed is the“Oligonucleotide Ligation Assay” (“OLA”) (Landegren et al., Science241:1077 1080 (1988)). The OLA protocol uses two oligonucleotides thatare designed to be capable of hybridizing to abutting sequences of asingle strand of a target. OLA, like LCR, is particularly suited for thedetection of point mutations. Unlike LCR, however, OLA results in“linear” rather than exponential amplification of the target sequence.

Still another method that may alternatively be employed is the “InvaderAssay” that uses a structure-specific flap endonuclease (FEN) to cleavea three-dimensional complex formed by hybridization of allele-specificoverlapping oligonucleotides to target DNA containing a singlenucleotide polymorphism (SNP) site. Annealing of the oligonucleotidecomplementary to the SNP allele in the target molecule triggers thecleavage of the oligonucleotide by cleavase, a thermostable FEN.Cleavage can be detected by several different approaches. Most commonly,the cleavage product triggers a secondary cleavage reaction on afluorescence resonance energy transfer (FRET) cassette to release afluorescent signal. Alternatively, the cleavage can be detected directlyby use of fluorescence polarization (FP) probes, or by massspectrometry. The invasive cleavage reaction is highly specific, has alow failure rate, and can detect zeptomol quantities of target DNA.While the assay traditionally has been used to interrogate one SNP inone sample per reaction, novel chip- or bead-based approaches have beentested to make this efficient and accurate assay adaptable tomultiplexing and high-throughput SNP genotyping.

Nickerson et al. have described a nucleic acid detection assay thatcombines attributes of PCR and OLA (Nickerson et al., Proc. Natl. Acad.Sci. (U.S.A.) 87:8923 8927 (1990)). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

Schemes based on ligation of two (or more) oligonucleotides in thepresence of a nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, arealso known (Wu et al., Genomics 4:560 569 (1989)), and may be readilyadapted to the purposes of the present invention.

In one embodiment, the presence or absence of an amplified DNA fragmentis indicative of the presence or absence of the trait itself or of aparticular allele of the trait. In one embodiment, a difference in thelength or nucleotide sequence of an amplified DNA fragment is indicativeof the presence of a particular allele of a trait, and thus enables todistinguish between different alleles of a trait.

In a specific embodiment of the invention simple sequence repeat (SSR)markers can be used to identify invention-relevant alleles in the parentplants and/or the ancestors thereof, as well as in the progeny plantsresulting from a cross of said parent plants. Simple sequence repeatsare short, repeated DNA sequences and present in the genomes of alleukaryotes and consists of several to over a hundred repeats of a givennucleotide motif.

Since the number of repeats present at a particular location in thegenome often differs among plants, SSRs can be analyzed to determine theabsence or presence of specific alleles.

In another embodiment of the invention SNP markers are used to identifyinvention-relevant alleles in the parent plants and/or the ancestorsthereof, as well as in the progeny plants resulting from a cross of saidparent plants

In the present invention a marker or a set of two or more markers may beused represented by a pair of PCR oligonucleotide primers comprisingforward primer of SEQ ID NO: 1 and reverse primer of SEQ ID NO: 2, orany adjacent marker that is statistically correlated and thusco-segregates with the Albugo candida resistance trait which primerslead to an amplification product in a PCR reaction exhibiting amolecular weight or a nucleotide sequence, which is essentiallyidentical or can be considered as an allele to that of a correspondingPCR amplification product obtainable from Brassica oleracea L vargemmifera UK 925, seed of which is deposited under deposit Number NCIMB41654 in a PCR reaction with the identical primer pair(s).

In a first step, DNA or cDNA samples are obtained from suitable plantmaterial such as leaf tissue by extracting DNA or RNA using knowntechniques. Primers that flank a region containing markers within theinvention-relevant qualitative trait locus disclosed herein before orwithin a region linked thereto, are then used to amplify the DNA sampleusing the polymerase chain reaction (PCR) method well-known to thoseskilled in the art.

Basically, the method of PCR amplification involves use of a primer or apair of primers comprising two short oligonucleotide primer sequencesflanking the DNA segment to be amplified or adapter sequences ligated tosaid DNA segment. Repeated cycles of heating and denaturation of the DNAare followed by annealing of the primers to their complementarysequences at low temperatures, and extension of the annealed primerswith DNA polymerase. The primers hybridize to opposite strands of theDNA target sequences. Hybridization refers to annealing of complementaryDNA strands, where complementary refers to the sequence of thenucleotides such that the nucleotides of one strand can bond with thenucleotides on the opposite strand to form double stranded structures.The primers are oriented so that DNA synthesis by the polymeraseproceeds bidirectionally across the nucleotide sequence between theprimers. This procedure effectively doubles the amount of that DNAsegment in one cycle. Because the PCR products are complementary to, andcapable of binding to, the primers, each successive cycle doubles theamount of DNA synthesized in the previous cycle. The result of thisprocedure is exponential accumulation of a specific target fragment,that is approximately 2<n>, where n is the number of cycles.

Through PCR amplification millions of copies of the DNA segment flankedby the primers are made. Differences in the number of repeated sequencesor insertions or deletions in the region flanking said repeats, whichare located between the flanking primers in different alleles arereflected in length variations or sequence of the amplified DNAfragments. These variations can be detected, for example, byelectrophoretically separating the amplified DNA fragments on gels or byusing capillary sequencer. By analyzing the gel or profile, it can bedetermined whether the plant contains the desired allele in a homozygousor heterozygous state or whether the desired or undesired allele isabsent from the plant genome.

In the alternative, the presence or absence of the desired allele may bedetermined by real-time PCR using double-stranded DNA dyes or thefluorescent reporter probe method.

Marker analysis can be done early in plant development using DNA samplesextracted from leaf tissue of very young plants or from seed. Thisallows to identify plants with a desirable genetic make-up early in thebreeding cycle and to discard plants that do not contain the desired,invention-relevant alleles prior to pollination thus reducing the sizeof the breeding population and reducing the requirements of phenotyping.

Further, by using molecular markers, a distinction can be made betweenhomozygous plants that carry two copies of the desired,invention-relevant allele at the Albugo candida resistance qualitativelocus and heterozygous plants that carry only one copy and plants thatdo not contain any copy of the favourable allele(s).

Thus, alternative markers can therefore be developed by methods known tothe skilled person and used to identify and select plants with an alleleor a set of alleles of a qualitative trait locus or loci according tothe present invention and as disclosed herein before.

For example, the nucleotide sequence of the amplification productobtained in PCR amplification using the a pair of PCR oligonucleotideprimers comprising forward primer of SEQ ID NO: 1 and reverse primer ofSEQ ID NO: 2 can be obtained by those skilled in the art and new primersor primer pairs designed based on the newly determined nucleotidesequence of the PCR amplification product. Furthermore, the markersaccording to the invention and disclosed herein before could bepositioned on a genetic map of a cultivated Brassica plant, inparticular species of the family Brassica oleracea, and known markersmapping in the same or homolog or ortholog region(s) could be used asstarting point for developing new markers.

Joint-analysis of genotypic and phenotypic data can be performed usingstandard software known to those skilled in the art. Plant introductionsand germplasm can be screened for the alleles at the correspondingAlbugo candida resistance locus disclosed herein, based on thenucleotide sequence(s) of the marker(s) at the marker locus/loci linkedto said Albugo candida resistance locus, and the molecular weight of theallele(s) using one or more of the techniques disclosed herein or knownto those skilled in the art.

The present invention therefore also relates to an isolated nucleic acid(preferably DNA but not limited to DNA) sequence that comprises a Albugocandida resistance locus of the present invention, or aresistance-conferring part thereof. Thus the markers discloses may beused for the identification and isolation of one or more markers orgenes from Brassica oleracea or other vegetable crops within the genusBrassica that are linked or encode Albugo candida resistance.

Also, in one embodiment, the present invention concerns a kit fordetection of Albugo candida resistance locus in Brassica oleracea, saidkit comprising a pair of PCR oligonucleotide primers able to amplify aDNA marker linked to the Albugo candida resistance locus.

In particular embodiment, in the said kit, the said DNA marker can beamplified in a PCR reaction by amplification of a DNA fragment with apair of PCR oligonucleotide primers represented by a forward primer ofSEQ ID No 1 and a reverse primer primer of SEQ ID No 2), and said DNAfragment comprises at least one SNP marker selected within the groupcomprising:

-   -   i. a SNP A represented by a T to C nucleotide exchange at        position 134 in the PCR amplified product,    -   ii. a SNP B represented by a C to T nucleotide exchange at        position 108 in the PCR amplified product,    -   ii. a SNP C represented by a T to C nucleotide exchange at        position 366 in the PCR amplified product.    -   In SNP A, C corresponds to resistant allele and T to susceptible        allele.    -   In SNP B, T corresponds to resistant allele and C to susceptible        allele.    -   In SNP C, C corresponds to resistant allele and T to susceptible        allele

More particularly, in a kit according to the present invention, the SNPA can be identified with a pair of PCR oligonucleotide primersrepresented by a forward primer of SEQ ID No 3 and a reverse primer ofSEQ ID No 4, and a DNA probe of SEQ ID No 5 defining the resistantallele. The present invention also provides a DNA marker that is linkedto Albugo candida resistance locus in Brassica oleracea and can beidentified in a PCR reaction by amplification of a DNA fragment with apair of PCR oligonucleotide primers represented by a forward primer ofSEQ ID No 1 and a reverse primer primer of SEQ ID No 2), said DNAfragment comprises at least one SNP marker selected within the groupcomprising:

-   -   i. a SNP A represented by a T to C nucleotide exchange at        position 134 in the PCR amplified product,    -   ii. a SNP B represented by a C to T nucleotide exchange at        position 108 in the PCR amplified product,    -   ii. a SNP C represented by a T to C nucleotide exchange at        position 366 in the PCR amplified product.

The invention also provides DNA marker wherein the SNP A can beidentified with a pair of PCR oligonucleotide primers represented by aforward primer of SEQ ID No 3 and a reverse primer of SEQ ID No 4 and aDNA probe of SEQ ID No 5 defining the resistant allele.

In one embodiment, the present invention also concerns the use of a DNAmarker according to the previous paragraphs for diagnostic selection ofAlbugo candida resistance locus in Brassica oleracea plant.

Also, the invention provides the use of DNA marker according to theprevious mentioned embodiments for identification in plant the presenceof Albugo candida resistance locus and/or for monitoring ofintrogression of the Albugo candida resistance locus in cultivatedBrassica oleracea plant.

In one embodiment the present invention discloses B. oleracea plantsresistant to Albugo candida and further resistant to clubroot, whereinthe resistance to clubroot is monogenic and dominant, including seedsand materials of said plants and the progeny thereof.

The present invention thus provides cultivated Brassica oleracea plantresistant to Albugo candida according to any of the embodimentsdescribed herein further characterized in that said plant resistant toclubroot disease, wherein the resistance to clubroot is monogenic anddominant

Brassica oleracea plants resistant to clubroot and method for obtainingthem are disclosed in EP1525317 entitled Clubroot resistant Brassicaplants.

The present invention also discloses methods to produce B. oleraceaplants resistant to Albugo candida and further resistant to clubroot,methods to transfer the clubroot resistance to clubroot susceptible orless resistant B. oleracea plants that are resistant to Albugo candida.

Albugo candida mainly affects leaves while and Plasmodiophra brassicaemainly attacks the roots of Brassica oleracea. It is thereforeadvantageous to get a Brassica oleracea plant having combined resistantagainst both pathogen in order to ensure an adequate growth of the plantfrom the roots to the leaves.

Higher yields may be obtained because of the absence of diseases onresistant plants. Moreover much less crop protection chemicals or nocrop protection chemicals at all are required against clubroot andagainst Albugo candida when B. oleracea plants of the present inventionare grown.

-   -   The present invention therefore discloses:

A B. oleracea plant resistant to clubroot disease, more particularly toclubroot disease caused by the pathogen Plasmodiophora brassicae andresistant to Albugo candida.

In a specific embodiment of the invention, the resistance to clubrootdisease is monogenic and dominant.

In another preferred embodiment, the B. oleracea plant that has combinedresistance against clubroot disease and against Albugo candida isbroccoli, white cabbage, cauliflower, Brussels sprouts, Borecole, Savoy,or red cabbage. In another preferred embodiment, the B. oleracea plantis homozygous or heterozygous for the resistance to clubroot. In anotherpreferred embodiment, the resistance to clubroot is genetically linkedto a molecular marker. Preferably, the molecular marker is obtainable byPCR amplification.

-   -   The present invention further discloses:

Seed of a Brassica oleracea plant which is resistant to Albugo candidaand resistant to clubroot disease, including the progeny thereof,wherein said seed or progeny comprises the resistances of the presentinvention.

In another preferred embodiment, said resistance to clubroot ismonogenic, preferably monogenic and dominant. In a preferred embodiment,the B. oleracea plant is homozygous for the clubroot resistance. Inanother preferred embodiment, the B. oleracea plant is heterozygous forthe clubroot resistance.

-   -   The present invention further discloses:

Seed of a plant disclosed above, including the progeny thereof, whereinsaid seed or progeny comprises the resistances against Albugo candidaand clubroot according to the present invention.

-   -   The present invention further discloses:

A method for producing a B. oleracea plant resistant to Albugo candidafurther comprising a monogenic and dominant resistance to clubrootcomprising the steps of:

a) providing a Brassica oleracea resistant to Albugo candida,

b) obtaining a B. rapa plant resistant to clubroot,

c) crossing said B. rapa plant with the B. oleracea plant resistant toAlbugo candida,

d) rescuing embryos resulting from the cross of step c),

e) regenerating a plant from a embryo of step d),

f) selecting a plant of step e) that is resistant to clubroot andresistant to Albugo candida,

g) back-crossing a plant resulting from step f) with a B. oleracea plantresistant to Albugo candida.

In a preferred embodiment, the method further comprises introgressingthe resistance into an elite B. oleracea inbred that is resistant toAlbugo candida. In another preferred embodiment, the method furthercomprises crossing said inbred to another B. oleracea inbred to producea hybrid

The foregoing description will be more fully understood with thereference to the following Examples. Such Examples are, however,exemplary methods of practising the present invention and are notintended to limit the scope of the invention.

The following Examples illustrate the invention:

EXAMPLE 1 Disease Tests Used to Test for the Presence of the Resistance

Resistance is not fully expressed in the cotyledons and therefore trueleaves (preferably 3-4 leaf stage) are used to inoculate plants. Plantsare sown in regular peat soil in the greenhouse in trays. After 7-10days after sowing, the seedlings are transplanted in 9×9×8 cm potsfilled with regular peat soil and grown for 3-4 weeks in a greenhouse atmoderate temperatures (18-20° C., night-day). When the first 3-4 leavesare fully grown, the plants can be inoculated.

Spores were collected with a vacuum pump from ripened pustules and drystored at −20° C. (Gilijamse et al., 2004). Sporangia were suspended incold demi-water and stored for 2 hr at 5° C. to allow the sporangia togerminate into zoospores. The zoospores were then sprayed onto the testplants (concentration 10⁻⁴-10⁻⁵ zoospores/ml) in a climate chamber with100% RH. Incubation during 10-14 d at 18-20° C. in a greenhouse afterwhich the white pustules start to appear. Final observation is doneusing a 1-9 scale in which 1=plants are fully covered with largepustules, and, 9=plants are healthy without showing any symptoms (Table1). Plants scoring 1-5 on this scale are regarded as being susceptibleand plants scoring 6-9 are classified as being resistant; 6-7 isregarded as intermediately resistant and 8-9 as standard resistant.

TABLE 1 Scale for scoring resistance against Albugo candida % leafcovered scale S/R Nb pustules with pustules 1 S >50 large pustules >80% 2 S 30-50 large pustules 50-80% 3 S 15-30 large pustules 20-50% 4 S 5-15large pustules  5-20% 5 S 1-5 large pustules and/or >20 small 1-5%pustules 6 R 5-20 small pustules 0.1-1%   7 R 1-5 small pustules <0.1%  8 R no pustules, only HR 0% 9 R no symptoms 0% S = susceptible, R =resistant

EXAMPLE 2 Transfer of the Albugo candida Resistance to B. oleracea

In 2001, individual plants of a Portuguese kale (HRI accession 12105,wild Brassica oleracea acephala; Couve Galega Frisada) were identifiedto have resistance against Albugo candida. These plants were selfed tofixate the resistance and then further back crossed with elite lines ofcabbage, B. sprouts, cauliflower and broccoli to introgress theresistance.

In Brussels sprouts, a parental line 1 was converted with Albugo candidaresistance by crossing with HRI 12105. After 3 generations of backcrosses the B3 segregated nicely 1:1 (Table 1, pool of 2 lines). After 4inbreeding cycles (B3F4) a near isogenic line (resembling phenotypicallyto Brussels sprouts parental line 1) was obtained with Albugo candidaresistance. This line was 100% resistant. Two test crosses were made,one gave 100% resistant plants the other 97% resistant plants.

This 100% resistant line was then crossed with a female line holding CMSin order to obtain the UK 925 Brassica oleracea L var gemmifera, seed ofwhich is deposited under deposit Number NCIMB 41654.

In another recurrent background line of Brussels sprouts, the B1segregated also nicely into 1:1 (Table 1).

Using a white cabbage background, a B1 revealed 1:1 segregation, afixated line showed 100% resistance as did the test F1. A B0 and B1 incauliflower segregated 1:1.

These examples are indicative for many more backcrosses in Brusselssprouts, cabbage, Savoy cabbage, broccoli and other cultivated Brassicaoleracea crops. Classifying the back crosses in Table 1, a S:R=1:1segregation is present (Chi-square, P>0.05) indicative for a single(semi-)dominant gene. The data also show that introgression intodifferent crops is possible in order to establish Albugo candidaresistance in different cultivated Brassica oleracea species such asBroccoli, cauliflower, white cabbage, savoy cabbage and Brusselssprouts.

TABLE 2 Disease results of back cross programs in Brussels sprouts (BS)and white cabbage (WC). Mean Plant generation S R % S % R Score Parentalline 1 (BS) F12 48 0 100 0 4.9 Parental line 1 with B3F4 0 48 0 100 8.1Albugo candida resistance Parent line (WC) F10 15 0 100 0 3.9 Fixatedline with B4F3 0 24 0 100 8.0 Albugo candida resistance Test F1 F1 0 240 100 7.8 Abrev: S = susceptible, R = resistant (score 6-9, see Table1). BS = Brussels sprouts, WC = White cabbage; Mean = mean of Albugocandida score (according Table 1). Similar experiments were done withCauliflower and Broccoli and showed that introgression of the Albugocandida resistance trait can be achieved by crossing and back-crossingwith resistance source in order to select and produce cultivatedBrassica oleracea plants with resistance to Albugo candida resistance.

EXAMPLE 3 Molecular Marker Development

Near isogenic lines (NILs) of white cabbage and Brussels sprouts withintrogression from HRI1215 Albugo candida resistance gene were used foridentification of Molecular Markers linked to resistance to Albugocandida disease.

To identify new markers linked to Albugo candida resistance fromHR11215, DNA of the near-isogenic lines (NILs) derived from a B3F3 werehybridized to the Brassica affymetrix array. The Arabidopsis homologs ofcandidate SFP with significantly different hybridization signals betweenthe resistant and susceptible NILs. The genomic positions of theArabidopsis homologs for a large number of candidates were clustered ina block of chromosome 5 that is syntenic to the region of B. oleraceachromosome 2 where Albugo candida resistance is located. Thesecandidates were sequenced on a trait-specific panel of fixed resistantand susceptible lines to identify polymorphisms for Taqman assaydevelopment. A homolog of At5g17610 carried SNP polymorphisms thatcorrelated with segregation for resistance in both Brussels sprouts andcabbage.

PCR Sequencing for SNP A, SNP B and SNP C.

Forward primer  (SEQ No 1) 5′ CACGACGTTGTAAAACGACAAGAGAATTGTGCGCTGC 3′Reverse primer  (SEQ No 2) 5′ CAGGAAACAGCTATGACCAAAAGCTGCCACGAACAC 3′

The set of primers amplify a sequence that contains SNPs at positions108 (SNP A), 134 (SNP B), and 366 (SNP C). The resistant and susceptiblesamples were genotyped by sequencing the PCR products obtained by theset of primers. The sequencing was done using a Sanger sequencingreaction followed by capillary electrophoresis.

Testing a verification panel thus revealed that the following SNPscosegregate with the resistance:

-   -   SNP A shows resistant allele with a C for T substitution at        position 134 in the amplified product; C corresponding to        resistant.    -   SNP B shows resistant allele with a T for C substitution at        position 108 in the amplified product; T corresponding to        resistant.    -   SNP C shows resistant allele with a C for T substitution at        position 366 in the amplified product; C corresponding to        resistant.        TaqMan Assay for SNP A

Taqman assay protocol has been developed to detect resistance to Albugocandida (white blister) introgressed from HR11215. This codominant assaycan be applied in Brassica oleracea breeding for the transfer andselection of Albugo candida resistance in broccoli, cauliflower,cabbage, Brussels sprouts, and bore kale

For SNP A a TaqMan assay was developed in order to allows a quickerdetermination of resistant vs susceptible individuals.

The linkage distance between the SNP A assay and the resistance locuswas estimated as 1.5 cM. The locus was mapped on chromosome 2.

Forward primer (SEQ No 3) 5′ CACCATCTAGGCTCTCCCCGAGC 3′ Reverse primer(SEQ No 4) 5′ GGAGCCAAGAATACAAATATTGTATGTAC 3′Probes:

(SEQ No 5) FAM - TCATGTTTCGTCCTAGTATAC - MGB - NFQ > Resistant specific(SEQ No 6) VIC - CTAATCATGTTTCGTTCTTC - MGB - NFQ > Susceptible specificAssay Conditions1. Isolate genomic DNA with standard DNA isolation protocol (Potassiumacetate) and resuspend in 100 μL of TE;2. Dilute template DNA to 1/30;3. Pipette 4 μL of each diluted DNA sample into individual wells;4. Cover and centrifuge the plate and place on ice;5. Make the mix the master mix

Initial Final Sigma protocol Volume (μL) concentration concentrationgDNA 4.00 Buffer 10x (Sigma) 1.00 10 X 1X MgCl₂ 1.20 25 mM 3 mM dNTP(2.5 mM each = 0.80 2.5 mM each 0.2 mM each 10 mM all) Sigma Taq 0.1322.5 U/μL 0.033 U/μl HiNK12509 = 0.10 10 μM 100 nM SO0005AA1FM HiNK12508= 0.10 10 μM 100 nM SO0005AA2VC HiNK12506 = 0.45 10 μM 450 nM SO0005AF1HiNK12507 = 0.45 10 μM 450 nM SO0005AR1 ROX 0.10 50X 0.5X H₂O QSP 1.668Total Volume 10.006. Load the plate on PCR machine;7. PCR program (on ABI Geneamp PCR 9700-384 plate format) as follows:

2 min 94° C. for Sigma Taq Polymerase 15 sec 94° C. {close oversizebrace} 40X  1 min 60° C.  5 min 72° C.8. Read the plate on ABI7900.

DEPOSIT

The following seed sample of Brassica oleracea L var gemmifera wasdeposited with NCIMB, Ferguson Building, Craibstone Estate, bucksbum,Aberdeen AB21 9YA, Scotland, UK, on Sep. 16, 2009 under the provisionsof the Budapest Treaty in the name of Syngenta Participations AG:

UK 925, deposit Number NCIMB 41654, deposition date 16 Sep. 2009.

What is claimed is:
 1. Cultivated Brassica oleracea plant resistant toAlbugo candida, comprising a resistance locus, such resistance locusbeing present in Brassica oleracea L var gemmifera UK 925, seed of whichis deposited under Deposit Number NCIMB 41654, and wherein saidresistance locus is a monogenic and semi-dominant resistance locuslocated on chromosome 2, and wherein the Albugo candida resistance locusis genetically linked to at least one marker locus, which co-segregateswith Albugo candida resistance trait and comprises a marker that can beidentified in a PCR reaction by amplification of a DNA fragment with apair of PCR oligonucleotide primers represented by a forward primer ofSEQ ID NO: 1 and a reverse primer of SEQ ID NO:
 2. 2. CultivatedBrassica oleracea plant resistant to Albugo candida according to claim1, wherein the resistance locus is a qualitative Albugo candidaresistance locus.
 3. Cultivated Brassica oleracea plant according toclaim 1 wherein the primer pair amplifies a DNA fragment that comprisesthe at least one marker locus which co-segregates with the Albugocandida resistance locus.
 4. Cultivated Brassica oleracea plantaccording to claim 3 wherein primer pair amplifies a DNA fragmentcomprising at least one SNP within the at least one marker locus whichco-segregates with the Albugo candida resistance locus.
 5. CultivatedBrassica oleracea plant according to claim 4 wherein the at least oneSNP is selected from the group consisting of: i. a SNP A represented bya T to C nucleotide exchange at position 134 in the PCR amplifiedproduct ii. a SNP B represented by a C to T nucleotide exchange atposition 108 in the PCR amplified product; and iii. a SNP C representedby a T to C nucleotide exchange at position 366 in the PCR amplifiedproduct.
 6. Cultivated Brassica oleracea plant according to claim 5wherein the SNP A can be identified with a pair of PCR oligonucleotideprimers represented by a forward primer of SEQ ID NO: 3 and a reverseprimer of SEQ ID NO: 4 and DNA probe of SEQ ID NO: 5 defining theresistant allele.
 7. Seed of cultivated Brassica oleracea plantaccording to claim 1 comprising the resistance locus contributing toresistance to Albugo candida.
 8. Method for producing a cultivatedBrassica oleracea plant, exhibiting resistance to Albugo candida,comprising the steps of: a. crossing the plant of claim 1 with acultivated Brassica oleracea plant, which is susceptible to Albugocandida or exhibits a low level of resistance against Albugo candida,and b. selecting progeny from said cross which exhibits Albugo candidaresistance and demonstrates association with said at least one markerlocus.
 9. Method according to claim 8, wherein the primer pair amplifiesa DNA fragment comprising at least one SNP within the at least onemarker locus which co-segregates with the Albugo candida resistancelocus.
 10. Method according to claim 9, wherein the at least one SNP isselected from the group consisting of: i. a SNP A represented by a T toC nucleotide exchange at position 134 in the PCR amplified product ii. aSNP B represented by a C to T nucleotide exchange at position 108 in thePCR amplified product; and iii. a SNP C represented by a T to Cnucleotide exchange at position 366 in the PCR amplified product. 11.Method for obtaining cultivated Brassica oleracea edible parts resistantto Albugo candida comprising the steps of i. sowing a seed of cultivatedBrassica oleracea according to claim 7, ii. growing said plant in orderto produce edible parts and harvesting the said edible parts produced bysaid plant.
 12. Method of protecting a field of cultivated Brassicaoleracea plants against infection by Albugo candida, wherein said methodis characterized by planting a seed according to claim
 7. 13. Method ofproducing hybrid seeds of a cultivated Brassica oleracea resistant toAlbugo candida comprising the steps of: i. planting a female Brassicaoleracea plant, and the male plant according to claim 1, ii. effectingcross pollination between both parents, iii. growing the plant till seedsetting, iv. collecting seeds, and v. obtaining the hybrid seeds.